NY-ESO-1 is one of the most immunogenic cancer testis antigens, able to induce strong humoral (antibody) and cellular (T cell) immune responses in patients with NY-ESO-1 expressing cancers either through natural or spontaneous induction by the patients tumor or following specific vaccinination using define peptides epitopes (Jager, et al., Proc. Natl. Acad. Sci. USA, 97(22):12198-12203 (2000)) or recombinant NY-ESO-1 protein (Davis, et al., Proc. Natl. Acad. Sci. USA, 101(29):10697-10702 (2004)). CD4+ T-cells play a critical role in generating and maintaining antigen specific cellular immune responses such as CD8+ T cells also referred as Cytotoxic T lymphocytes (CTL's) and humoral immune responses such as antibodies. The identification and characterization of additional NY-ESO-1 peptide epitopes presented by MHC class II molecules which may be recognized by naturally induced or vaccine induced CD4+ T cells remains vital for the design, development and evaluation of cancer vaccines utilizing the NY-ESO-1 antigen.
In the disclosure which follows CD4+ T-cells form 3 cancer patients with NY-ESO-1 expressing tumors were stimulated with synthetic overlapping 18 mer peptides spanning the entire sequence of NY-ESO-1. Two novel peptide epitopes for NY-ESO-1 were identified that were recognized by CD4+ T-cells from the 3 patients. Antigen specific CD4+ T-cell clones were generated by repetitive stimulation with the novel peptide epitopes, NY-ESO-1 p49-66 and p55-72. Further experiments showed that the NY-ESO-1 p49-80-region contains at least two different CD4+ T-cell epitopes. Partially histocompatible EBV-B-cell lines and MHC class II specific blocking antibodies, were used to determine that these epitopes were presented in the context of the MHC class II molecule HLA-DQ B1 03011(DQ7). Natural processing and presentation of these epitopes was indicated by NY-ESO-1 specific CD4+ T cell recognition of an EBV-B-cell line expressing NY-ESO-1 and by T cell recognition of dendritic cells (DC's) exogenously loaded with NY-ESO-1 protein or infected with recombinant adenovirus-NY-ESO-1 (Ad-ESO) construct. Detectable IFN-γ and TNF-α cytokine production by the CD4+ T cells and lack of IL-4 secretion indicated that the T-cell clones belonged to the Th1 subtype, the subset of T cells that play a key role in the induction of specific CD8+ T-cell immunity. These new MHC (HLA) class II restricted NY-ESO-1 epitopes represent important tools to monitor spontaneous as well as peptide, protein or viral vector vaccine-induced immune responses in patients with NY-ESO-1 positive tumors or as a component of an NY-ESO-1 peptide cancer vaccine.